North American Upper Mid-West iGEM Meet-Up
Collaboration with Purdue University
Shared protocol and sent the Purdue iGEM team our bacterial strains and assembled bioreactors to promote repeatability in science:
Connected Purdue team to a professor at MSU for use of a GC/Mass Spectrometry instrument
Quantification of Benzene via GC/Mass Spectrometry
- Dates of Collaboration: 10/7/17-10/16/17
- Where: GC/Mass Spec Facility- MSU
- -Dr. Dan Jones
Preparing Phage Lysate
- Purpose- The overall genetic construct used to degrade benzene into acetyl-coA and pyruvate consists of 9 enzymes, and was broken into two plasmids to increase the success rate of cloning. Purdue only has been able to clone the first of these two plasmids into E. coli, Upper Operon (UO). The upper operon expresses several enzymes pertaining to benzene degradation, however it does not express the protein transporter necessary for cellular benzene uptake. To get around this obstacle, you will test the induced cellular lysate instead of the living culture via GC.
At the end of this protocol, you should have 30mL of uninduced cellular lysate and 30mL of induced cellular lysate.
- aTc (inducing agent)
- 2 autoclaved 125ml Erlenmeyer flasks
- LB broth
- Incubator with shaker
- Spectrophotometer (with cuvettes if necessary)
1. Inoculate a culture of UO E. coli in LB with chloramphenicol, CM, (25mg/L) from the provided stock culture as well as a culture of wild type E. coli and incubate at 37°C while shaking at approximately 250rpm overnight
- (25mg)(1L/1000mL)(1mL/30mg)(10mL)= 0.0083333mL=> 8.33uL of MSU’s 30mg CM
- 10mL LB + 50uL culture+ 8.33uL CM
- *CM is to only be added to UO, NOT the wild type
- In an autoclaved 125ml Erlenmeyer flask, aseptically dispense the following:
- 35mL LB broth
- Chloramphenicol to achieve 25mg/L
- .75mL UO culture from the previous day
- In another autoclaved 125ml Erlenmeyer flask, aseptically dispense the following:
- 35mL LB
- 1.75ml wild type E. coli culture from the previous day
- CM (ONLY FOR UO-- to acheive 25mg/L)
- For the Wild Type E. coli (WT) only: Incubate overnight at <35°C while shaking at approximately 250rpm
- For the UO only: Incubate at 37°C while shaking at approximately 250rpm for 1.5-2.5 hours. After 1.5 hours, check the OD600 periodically until an absorbance of 0.4-0.6 is obtained
When growing to OD for UO….
After the first 1.5 hours: OD was 0.529
- Measure the OD600 of both cultures (you will need to dilute a small volume from each sample, as absorbance does not follow a linear regression when OD600 > 1) Note: The cultures were around an OD of .980
- Aliquot out the equivalent of 10mL of cells (of both cultures) of OD600 = 1 into individual 15mL conical tubes (in triplicate, 6 tubes total)
- Centrifuge at top speed for 5 minutes
- After centrifugation, cells were resuspended in 9mL 1M Tris-HCl (pH 7.5) and then transferred to 50mL centrifuge tubes
- Of each culture (UO and WT) there was one 50mL centrifuge tube and 50mL of Tris was added in total to the cultures/resuspended to ensure more vials for sampling Note: The reason this was done was to ensure that benzene would not evaporate from multiple punctures in the caps; therefore, there were multiple samples to test of each culture/plasmid. Lyse cells via sonication Note: This was not done due to concerns from Dr. Jones regarding the activity of enzymes and whether or not they would metabolize Benzene after sonification. Also, there was not much notice on the use of Sonification in order to sign up ahead of time to use the machine and to make sure to have access to the building.
- Add 1mL 50 mM NADH per tube (will be provided) 500uL added to each sample/tube (refer to the Quantification of Benzene protocol below)
- Purpose: As a metric in determining the effectiveness of the modified E. Coli to degrade Benzene, it is necessary to quantify and measure the rate at which benzene disappears from a closed system.
- Phase I: GC Optimization.
- Prepare Standard Solutions
- For each standard, mix in a 20mL headspace vial and seal
- The samples will be tested at 125ug/mL of benzene, so the following standards should be prepared by adding benzene to 10mL of Tris/HCl pH 7.5 broth: Blank
- 20ug/mL – 0.23uL pure benzene
- 50ug/mL – 2.29uL pure benzene
- 80ug/mL – 3.66uL pure benzene
- 110ug/mL – 5.04uL pure benzene
- 140ug/mL – 6.41uL pure benzene
- Prepare Cultures for Headspace Extraction
- In a 20 mL headspace vial add:
- 5.0 mL of culture (WT and UO)
- 2.86uL of pure benzene (WT and UO)
- 500uL NADH
- Using an autosampler, determine the amount of benzene in each headspace vial every 2-6 hours for 1-2 days
Skype call with our collaborators at University of Purdue
Skype call with our collaborators at Universities of Cologne and Duesseldorf in Germany.